Objective To construct the eukaryotic expression vector of PRDX3 labeled with FLAG tag and to study its localization in human tongue cancer cell line SCC15.Methods PRDX3 gene was obtained from the breast library by PCR and cloned into PCDH vector to construct PCDH-FLAG-PRDX3.The plasmid was transiently transfected into 293T cells and the expression was detected by Western blot.Subcellular localization was detected by cellular immunofluorescence.Results The result of double digestion and sequencing showed that PCDH-FLAG-PRDX3 eukaryotic expression vector was constructed.The expression of FLAG-PRDX3 in human 293T cells was positively confirmed by Western blotting.In human tongue cancer cell line SCC15, the result of cellular immunofluorescence showed FLAG-PRDX3 was located in the cytoplasm rather than in the nucleus.Conclusion PRDX3 eukaryotic expression vector labeled with FLAG tag is constructed successfully, which is located in cytoplasm in human SCC15 cells.Construction and identification of PRDX3 could shed light on the function and mechanism of PRDX3 in tongue cancer.

Objective To explore genes expression of adiponectin receptors during differentiation of SW872 preadipocytes. Met-(hods) SW872 preadipocytes were cultured in vitro and induced to differentiate by 0.6 mmol/L oleic acid. During the progress of diffe-(rentiation), the morphological changes of SW872 cells were observed and the differentiation rate was assayed by oil-red O staining. Adiponectin receptors mRNA was measured by reverse transcription-polymerase chain reaction during differentiation of SW872 preadipocytes. Results 1.After stimulated by 0.6 mmol/L oleic acid for 72 hours, almost all SW872 cells were differentiated,and there were lots of fat droplets in the cells.2.There were adiponectin receptors genes expressions in SW872 preadipocytes.After 72 hours,and the levels of adiponectin receptor(AdipoR) 1 mRNA and AdipoR 2 mRNA were markedly increased up to 2.54 and 4.09 times,respectively. Conclusion There are AdipoR1 and AdipoR2 genes expressions in fat cells and the expressions are differentiation-dependent.

Objective To study the effect of oleic acid on the differentiation of SW872 preadipocytes.Methods SW872 prea-(dipocytes) were cultured and induced to differentiate by 0.6 mmol/L oleic acid in vitro.After 24 h,48 h and 72 h of differentiation,the morphological changes of SW872 preadipocytes were observed and the differentiation rate was assayed by oil-red O staining.In addition,triglyceride(TG) mass was detected by chemical colorimetry methods.During the differentiation of SW872 preadipocytes,transcription factors including peroxisome proliferator activated receptor-?_2(PPAR-?_2) and CAAT/enhancer binding protein-?(C/EBP-?)(mRNA) were also measured by reverse transcription-polymerase chain reaction(RT-PCR).Results 1.SW872 preadipocytes were fibroblastic and had no obvious fat droplet in cytoplasm.However,when stimulated for 72 hby 0.6 mmol/L oleic acid,SW872 preadipocytes became more bigger and rounder and differentiated into mature adipocytes with lots of fat droplets in the cells.2.Compared with that of predifferentiation,the concentration of TG mass increased by 14 folds after 72-hour differentiation(P

AIM: To construct human Egr-1 promoter luciferase reporter system and study its activity induced by i-onizing radiation. METHODS: Egr-1 promoter was obtained by human genomic PCR and cloned into pGL3-basic vector. After transfection of recombinant plasmid into human tumor cells, the Egr-1 promoter activity induced by ionizing radiation was detected by luciferase reporter assay. RESULTS: The luciferasy reporter system of Egr-1 promoter was successfully constructed. The activity of Egr-1 promoter was substantially increased after different doses of IR and reached to the peak at the time point of 48h after IR. CONCLUSION: The Egr-1 promoter was constructed in this study showed IR inducible activity in tumor cells, laying foundation for the research of radiation, mediated gene therapy.

Objective To construct the eukaryotic expression vector of human telomerase RNA component ( hTR) and study its biological function tentatively .Methods hTR Gene was obtained by PCR from cDNA template , which was reverse transcribed from 293T mRNA and cloned into pCDNA3.0 vector.The recombinant plasmid and empty vector were trans-fected into 293T cells, and hTR expression was identified by qRT-PCR.HepG2 cells that stably transfected with pCDNA3.0-hTR were constructed and identified by qRT-PCR.These cells were used to assess the interaction of hTR with human telomerase revese transcriptase ( hTERT ) and dyskerin .Telomerase activity was also detected in HepG 2 cells transfected with pCDNA3.0-hTR.Results pCDNA3.0-hTR eukaryotic expression vector was successfully constructed by double digestion identification .The inserted fragment was confirmed by sequencing .The expression of hTR in human 293T cells and HepG2 pCDNA3.0-hTR stable cell line was identified.In addition, qRT-PCR and Western blotting results showed that hTR could interact with hTERT and dyskerin , while hTR overexpression could not regulate the telomerase activity in HepG2 cells.Conclusion The eukaryotic expression vector of pCDNA 3.0-hTR is successfully constructed and expressed.This study will contribute to the further study of cancer therapy targeting hTR .

Objective To construct eukaryotic expression vector of wild type E 4F1 and the mutant deleting amino acid region 32-81, and to detect the interaction between wild type or mutant E 4F1 and p53 and to study the effect of E4F1 on the expression level of p21.Methods Wild type and mutant sequences of E 4F1 were amplified from the mammary library using standard PCR and recombinant PCR .The sequences were cloned into pXJ 40-MYC vector to generate the MYC-E4F1 and MYC-E4F1(Δ32-81) recombinant plasmids that were transfected into 293T cells and identified by Western blotting . FLAG-p53 and MYC-E4F1 or MYC-E4F1(Δ32-81) were co-transfected into 293T cells and immunoprecipitation assay was performed to detect the interaction of wild type or mutant E 4F1 with p53.Wild type and mutant E4F1 expressing vec-tors were co-transfected into osteosarcoma U2OS cells and the expression of p21was detected.Results Recombinant plas-mids of MYC-E4F1 and MYC-E4F1(Δ32-81) were successfully constructed.Both wild type and mutant E4F1 interacted with p53.Deletion of amino acid region 32-81 of E4F1 increased the interaction .The expression level of p21 was in-creased by wild-type E4F1, but not by mutant E4F1.Conclusion The eukaryotic expression vector of wild type E4F1 and its deletion mutant is successfully constructed .Both of them interact with p53.Deletion of amino acid region 32-81 of E4F1 increases the interaction .This study contributes to further studies on the regulation and mechanism of E 4F1 on p53.

OBJECTIVEIn this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NF-κB) in modulating adhesion molecules expressions.

METHODSNeutrophils and BEAS-2B cells (one human bronchial epithelial cell line) were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction (real-time qPCR). Phosphorylated p38 MAPK and inhibitor κB were analyzed by Western blot.

RESULTSIn co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrophils were enhanced significantly (P<0.05). Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor κB in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin (P<0.05).

CONCLUSIONSCoculture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF-κB pathways, and exhibiting its anti-inflflammation activity.

Animals ; Base Sequence ; Bronchi ; cytology ; enzymology ; metabolism ; Cattle ; Cell Adhesion Molecules ; metabolism ; Cell Line ; Coculture Techniques ; DNA Primers ; Down-Regulation ; drug effects ; Epithelial Cells ; enzymology ; metabolism ; Isoflavones ; pharmacology ; NF-kappa B ; metabolism ; Neutrophils ; enzymology ; metabolism ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; p38 Mitogen-Activated Protein Kinases ; metabolism

Objective To explore the genotype of patients with glucose-6-phosphate dehydrogenas(G6PD)deficiency in Dong-guan and provide the basis for the clinical diagnosis and prevention.Methods The clinical data of patients who took G6PD activity screening in the hospital were collected from January 2011 to December 2013,the G6PD/6PGD ratios were recorded.469 patients with positive G6PD/6PGD ratio were randomly enrolled in the study,whose mutations were detected by reverse dot blot(RDB)as-say.Results During this period,we measured G6PD activity of 16 464 cases by G6PD/6PGD ratios,there were 672 positive cases, the positive rate was 4.08%.Randomly selected 469 positive samples,detected their genotye by RDB assay.We detected 173 cases of G1376T,141 cases of G1388A,82 cases of A95G,60 cases of G871A,23 cases of G392T,14 cases of C1024T.In addition to that, we also found some rare mutations,such as 6 cases of C1004T,2 cases of T517C,1 cases of C1360T.65 cases of C1311T gene poly-morphism and 96 cases of dual gene mutations were detected.Conclusion The incidence of G6PD deficiency is high and the gene mutation types in Dongguan are both representative for Chinese population and with local heterogeneity.The study on gene muta-tions of G6PD deficiency is benefit for diagnosis and prevention.

Objective To construct PES1 shRNA stable expression cell lines in tongue squamous cell carcinoma ( TSCC) cells and to study the effect of knockdown of PES 1 on the growth of TSCC cells .Methods Recombinant lentivirus carrying PES1 shRNA was packaged and obtained in 293T cells.TSCC cells (Tca8113, SCC6 and SCC15) were infected with the lentivirus and selected for stable cells .PES1 expression was identified by Western blot .The effect of inhibition of PES1 on the growth and cell cycle of TSCC cells was detected by growth curve and flow cytometry .Results TSCC cells stably expressing PES1 shRNA were constructed.Knockdown of PES1 inhibited cell proliferation and induced cell cycle ar-rest at G0/G1 phase.Knockdown of PES1 inhibited expression of cyclin D1 in TSCC cells.Conclusion Inhibition of PES1 results in reduced cell proliferation , cell cycle arrest at G 0/G1 phase and reduction of cyclin D 1 expression in TSCC cells . PES1 may be a target for TSCC gene therapy .

BACKGROUND: It is still a problem to achieve early and accurate diagnosis of Alzheimer's disease, and delayed treatments often occur in a large number of patients because of late diagnosis or misdiagnosis. Therefore, the development and improvement of related bioanalytical methods are of great importance for the biomarker detection of Alzheimer's disease which is still lack of means that are sensitive, efficient and low-cost. OBJECTIVE: To summarize the diagnostic methods and related biomarkers of Alzheimer's disease, and to sum up the research progress in novel biomimetic receptors for the early detection of Alzheimer's disease biomarkers. METHODS: PubMed, CNKI and Wanfang databases were used to search articles related to the biomarker studies of Alzheimer's disease and relevant studies about methods of biomarkers detection published from 2000 to 2016. The key words were "Alzheimer's disease, biomarkers, detect/detecting/detection" in Chinese and English, respectively. Finally 40 articles were obtained for the review. RESULTS AND CONCLUSION: At present, the bioanalytical methods used for biomarker detection of Alzheimer's disease mostly utilize antibodies as recognition and capture elements of biomarkers, but there are some limitations using traditional antibodies as detection receptors. Thus, novel biomimetic receptors can be substituted for conventional antibodies. Novel biomimetic receptors have high specificity, small size, low production costs and high product stability, and their chemical modification process is relatively convenient. Biomimetic receptors developed for protein analysis include aptamers, polypeptide receptors, peptoid receptors, molecularly imprinted polymers, nanobodies, gelsolin and cucurbit urils. Detection of biomarkers with novel biomimetic receptors instead of conventional antibodies will be more accurate and timely in the early diagnosis of Alzheimer's disease.